Information on population age structure of Anopheles gambiae mosquitoes under natural conditions is fundamental to the understanding of vectorial capacity, and it is crucial for assessing the impact of vector control measures on malaria transmission. Control measures such as insecticide-treated bed nets not only reduce malaria vector abundance, but they also very likely reduce the life span of mosquitoes. New biological insecticides are found to preferentially kill older mosquitoes in the laboratory. Evaluation of the effects of the new biological insecticides on mosquito populations in nature requires accurate methods for An. gambiae age determination. Although age structure is a key parameter in mosquito ecology and malaria transmission, existing An. gambiae age-grading methods, based on morphological changes in the reproductive system, external marks and somatic changes with aging such as cuticular hydrocarbons, are either not sufficiently reliable or not sensitive for ecological and epidemiological studies. Recently, gene transcriptional profiles were found to be sensitive and reliable for age grading in Aedes aegypti. In order to adapt this new technique to An. gambiae, age-grading calibration models specifically for An. gambiae must be established, and the accuracy and generalizability of the method must be established. Accordingly, the following two specific aims are developed. First, we will adapt the gene-expression based age prediction method that has been very successful in Ae. aegypti to An. gambiae by identifying appropriate marker genes, establishing calibration models and examining the accuracy and reliability of the methods for An. gambiae age grading. Multiplex real-time PCR assays will be developed as an important means of reducing the costs associated with the new age-grading method. This aim will also test the hypothesis that the gene expression-based age-grading method offers a more precise estimation of An. gambiae age structure than the traditional ovarian tracheation method. Second, we will apply the new age-grading technique to test the hypothesis that the use of insecticide-treated bednets causes a significant shift of An. gambiae population age structure towards young individuals in western Kenya. An important outcome of this project is that we will establish new methods for An. gambiae age grading that are applicable to natural populations and therefore, the project will provide a very valuable tool to the scientific community for ecological and behavioral research of African malaria vectors. PUBLIC HEALTH RELEVANCE How long a mosquito lives in natural environment is not only a curious question to school children and general public, but it is also an important determinant of the ability of a mosquito to transmit diseases and infection risks. However, existing methods for determining An. gambiae mosquito age are either not sufficiently reliable or not sensitive for ecological and epidemiological studies. Recent studies in Aedes aegypti found that gene transcriptional profiles are excellent markers for age grading of the yellow fever mosquitoes. This application will adapt the gene expression-based age grading technique to Anopheles gambiae mosquitoes, the most important malaria vector in Africa. The proposed research will provide a valuable tool for better understanding mosquito age structure, a key parameter in mosquito ecology and malaria epidemiology.